Oligomerization and nanocluster organization render specificity

Nanoclusters are anchored to membranes, either within them or in the cytoplasm latched onto the cytoskeleton, whose reorganization can regulate their activity. Nanoclusters have been viewed in terms of cooperativity and activation; here we perceive nanocluster organization from a conformational standpoint. This leads us to suggest that while single molecules encode activity, nanoclusters induce specificity, and that this is their main evolutionary aim. Distinct, isoform‐specific nanocluster organization can drive the preferred effector (and ligand) interactions and thereby designate signalling pathways. The absence of detailed structural information across the nanocluster, due to size and dynamics, hinders an in‐depth grasp of its mechanistic features; however, available data already capture some of the principles and their functional ‘raison d'être’. Collectively, clustering lends stability and reduces the likelihood of proteolytic cleavage; it also increases the effective local concentration and enables efficient cooperative activation. However, clustering does not determine the ability of the single molecule to function. Drugs targeting nanoclusters can attenuate activity by hampering cooperativity; however, this may not perturb activation and signalling, which originate from the molecules themselves, and as such, are likely to endure. What then is the major role of nanoclustering? Assuming that single molecules evolved first, with a subsequent increase in cellular complexity and emergence of highly similar isoform variants, evolution faced the threat of signalling promiscuity. We reason that this potential risk was thwarted by oligomerization and clustering; clustering confers higher specificity, and a concomitant extra layer of cellular control. In our Ras example, signalling will be more accurate as a dimer than as a monomer, where its isomer specificity could be compromised.     

Oligomerization of the human ABC transporter ABCG2: evaluation of the native protein and chimeric dimers

Human ABCG2, a member of the ATP binding cassette (ABC) transporter superfamily, is overexpressed in numerous multidrug-resistant cells in culture. Localized to the plasma membrane, ABCG2 contains six transmembrane segments and one nucleotide binding domain (NBD) and is thought to function as a dimer or higher order oligomer. Chimeric fusion proteins containing two ABCG2 proteins joined either with or without a flexible linker peptide were expressed at the plasma membrane and maintained drug transport activity. Expression of an ABCG2 variant mutated in a conserved residue in the Walker B motif of the NBD (D210N) resulted in a non-functional protein expressed at the cell surface. Expression of an ABCG2 chimeric dimer containing the D210N mutation in the first ABCG2 resulted in a dominant-negative phenotype, as the protein was expressed at the surface but was not functional. Using a bifunctional photoaffinity nucleotide analogue and a non-membrane-permeable cysteine-specific chemical cross-linking agent, a dimer is the predominant form of oligomerized ABCG2 under our assay conditions. Furthermore, these experiments demonstrated that the dimer interface includes, but may not be limited to, interactions between residues in each monomeric NBD and separate disulfide interactions between the cysteines in the third extracellular loop of each monomer. By changing all three extracellular cysteines to alanine, we showed that although extracellular disulfide bonds may exist between monomers, they are not essential for ABCG2 localization, transport activity, or prazosin-stimulated ATPase activity. Together, these data suggest that ABCG2 functions as a dimer, but do not exclude functional higher order oligomers.     

Amphotericin B covalent dimers bearing a tartarate linkage

Amphotericin B (AmB, 1) is known to assemble together and form an ion channel across biomembranes, by which the drug presumably exerts its antimicrobial activity. To access the whole architecture of this channel assemblage, the understanding of binary interaction between AmB molecules is of prime importance because the dimeric interaction is the basis of the assemblage. In this context, we have recently reported covalently conjugated AmB dimers such as 2 and 3 with a long linker, which show prominent hemolytic potency and ion-channel activity. To evaluate the effect of the length and hydrophilicity of linker parts on the activity, we prepared new dimers bearing tartarate linkages (4 and 5). Especially, 5 exhibited potent hemolytic activity (EC50, 0.03 microM) surpassing those of AmB, 2, and 3. Measurements of UV and CD spectra of 5 in liposomes indicated that AmB portions of 5 could adopt appropriate arrangements in molecular assemblage in spite of the short linkage, and also indicated that the assemblage formed by 5 appeared more stable than AmB. These short-tethered dimers are expected to be a promising tool to reveal the mechanism of dimeric interaction in the ion channel formed by AmB.     

Facilitated Oligomerization of Mycobacterial GroEL: Evidence for Phosphorylation-Mediated Oligomerization

The distinctive feature of the GroES-GroEL chaperonin system in mediating protein folding lies in its ability to exist in a tetradecameric state, form a central cavity, and encapsulate the substrate via the GroES lid. However, recombinant GroELs of Mycobacterium tuberculosis are unable to act as effective molecular chaperones when expressed in Escherichia coli. We demonstrate here that the inability of M. tuberculosis GroEL1 to act as a functional chaperone in E. coli can be alleviated by facilitated oligomerization. The results of directed evolution involving random DNA shuffling of the genes encoding M. tuberculosis GroEL homologues followed by selection for functional entities suggested that the loss of chaperoning ability of the recombinant mycobacterial GroEL1 and GroEL2 in E. coli might be due to their inability to form canonical tetradecamers. This was confirmed by the results of domain-swapping experiments that generated M. tuberculosis-E. coli chimeras bearing mutually exchanged equatorial domains, which revealed that E. coli GroEL loses its chaperonin activity due to alteration of its oligomerization capabilities and vice versa for M. tuberculosis GroEL1. Furthermore, studying the oligomerization status of native GroEL1 from cell lysates of M. tuberculosis revealed that it exists in multiple oligomeric forms, including single-ring and double-ring variants. Immunochemical and mass spectrometric studies of the native M. tuberculosis GroEL1 revealed that the tetradecameric form is phosphorylated on serine-393, while the heptameric form is not, indicating that the switch between the single- and double-ring variants is mediated by phosphorylation.GroEL, an essential chaperonin, is known to form a ring-shaped structure for sequestering substrate proteins from the crowded cellular milieu and is responsible for the occurrence of various cellular processes, such as de novo folding, transport, and macromolecular assembly, within a biologically relevant time scale (7, 26, 48, 53). In Escherichia coli, GroEL, along with its cofactor GroES, assists the folding of about 10 to 30% of cytosolic proteins, among which some are known to be essential for cell viability (15, 26, 27, 31). GroEL was originally identified as the host factor responsible for phage λ and T4 capsid protein assembly and was subsequently shown to be essential for cell viability (17, 20). E. coli groEL is found in an operonic arrangement with groES (groESL), and its expression is regulated by multiple promoter elements.GroEL function has been shown to be a complex interplay between its interaction with and encapsulation of substrate proteins, with concomitant conformational changes induced by ATP binding, hydrolysis, and GroES binding (24, 56, 62). E. coli GroEL exists as a homotetradecamer forming two isologous rings of seven identical subunits each. Crystallographic analyses have delineated the three-domain architecture of GroEL monomers and the GroES-GroEL interactions (4, 63). The central region of the GroEL polypeptide, spanning amino acid residues 191 to 376, constitutes the GroES and substrate polypeptide-binding apical domain. The equatorial ATPase domain spanning two extremities of the GroEL polypeptide, that is, residues 6 to 133 and 409 to 523, is responsible for the ATPase activity and the bulk of intersubunit interactions. The hinge-forming intermediate domain, spanning two regions on the polypeptide, namely, residues 134 to 190 and 377 to 408, connects the said two domains in the tertiary structure. The conformational changes resulting from ATP binding and hydrolysis at the equatorial domain are coupled to those occurring at the apical domain via this hinge region (4, 63).The usual size limit for the substrate proteins, as shown by both in vitro and in vivo studies, is around 57 kDa, although the cis cavity is reported to theoretically accommodate larger proteins, on the order of 104 kDa (10, 27, 35, 46). Productive in vivo folding of the proteins larger than the usual size limit, such as the 86-kDa maltose binding protein fusion and 82-kDa mitochondrial aconitase, has also been reported (9, 29). Since such large substrates are difficult to accommodate in the central cavity, it has been suggested that their productive folding might occur outside the cis cavity. These studies therefore indicate that the substrate recognition patterns of GroEL may be more diverse than initially thought.Recent genome annotation studies of various bacteria have revealed that a few bacterial genomes possess multiple copies of groEL genes (2, 18, 30). The Mycobacterium tuberculosis genome bears two copies of groEL genes (groELs). One of these, groEL1, is arranged in an operon, with the cognate cochaperonin groES being the first gene, while the second copy, groEL2, exists separately on the genome (13). Recombinant mycobacterial GroELs were shown to possess biochemical features that deviated significantly from the trademark properties of E. coli GroEL. The most striking feature of M. tuberculosis GroELs, however, was their oligomeric state, where contrary to expectations, in vitro they did not form the canonical tetradecameric assembly when purified from E. coli. The proteins instead existed as lower oligomers (dimers) irrespective of the presence or absence of cofactors, such as the cognate GroES or ATP (40, 41). Furthermore, they displayed weak ATPase activities and GroES independence in preventing aggregation of the denatured polypeptides.Evolutionary studies of M. tuberculosis groEL sequences have suggested rapid evolution of the groEL1 gene, yet without turning these into pseudogenes (21). The other hypothesis suggests that M. tuberculosis, being an organism that grows slowly, might require GroEL function that does not utilize ATP rapidly but, rather, with a slow turnover rate. Alternately, additional mechanisms might exist in M. tuberculosis which could mediate regulated oligomerization of M. tuberculosis chaperonins. Such regulation might help in the controlled utilization of ATP in nutrient-deprived M. tuberculosis, as observed for other chaperones, such as small heat shock proteins (23).In the present study, we have exploited the unusual oligomeric status of the recombinant M. tuberculosis GroELs to study the significance of oligomer formation for GroEL''s function as a molecular chaperone. Furthermore, we have explored the possibility of the existence of regulated oligomerization for native M. tuberculosis GroELs in their natural setting. We first show that M. tuberculosis groEL genes are not capable of complementing a conditional allele of E. coli groEL, namely, groEL44. The results of phenotypic and biochemical analyses of GroEL variants obtained by gene shuffling and domain swapping suggest that the impaired chaperoning ability of recombinant M. tuberculosis GroELs is a consequence of their inability to form higher-order oligomers in E. coli and that oligomerization is the prelude to the formation of an active GroEL chaperonin. Further, by immunochemical and mass spectrometric (MS) analysis of native mycobacterial GroELs, we show that M. tuberculosis GroEL1 exists in multiple oligomeric forms, viz., monomeric, dimeric, heptameric (single ring), and tetradecameric (double ring) forms, and that the switch between single-ring and double-ring variants is operated by phosphorylation on a serine residue. These observations suggest that the determinants of oligomerization for M. tuberculosis GroEL1 are distinct from those of its E. coli counterpart and that it does oligomerize in M. tuberculosis (its native environment), whereas it loses its oligomerization capability when expressed in E. coli. It could thus be possible that M. tuberculosis GroEL1 requires a certain native M. tuberculosis protein, probably a eukaryotic-like Ser-Thr protein kinase, to oligomerize properly, though the precise reason cannot be discerned by these observations.     

Oligoaminonucleoside phosphoramidates. Oligomerization of dimers of 3''-amino-3''-deoxy-nucleotides (GC and CG) in aqueous solution.

The self-complementary 5'-phosphorylated dinucleoside 3' (N)----5' (P)-linked phosphoramidates with sequence GC (8a), CG (8b) and the tetranucleoside triphosphoramidate with sequence GCGC (10a) and CGCG (10b) have been synthesized and characterized by physicochemical and enzymatic methods. The dinucleosides 8a or 8b oligomerize in aqueous solution in the presence of a water-soluble carbodiimide. This process is efficient and regiospecific. In the case of GC it produces alternating 3' (N)----5' (P)-linked phosphoramidates up to 15 dimeric units in length with a yield in excess of 70%. The oligomerization of the CG isomer is much less efficient. The mechanism of oligomerization is discussed.     

RNA-ligand interactions: affinity and specificity of aminoglycoside dimers and acridine conjugates to the HIV-1 Rev response element

Semisynthetic aminoglycoside derivatives may provide a means to selectively target viral RNA sites, including the HIV-1 Rev response element (RRE). The design, synthesis, and evaluation of derivatives based upon neomycin B, kanamycin A, and tobramycin conjugates of 9-aminoacridine are presented. To evaluate the importance of the acridine moiety, a series of dimeric aminoglycosides as well as unmodified "monomeric" aminoglycosides have also been evaluated for their nucleic acid affinity and specificity. Fluorescence-based binding assays that use ethidium bromide or Rev peptide displacement are used to quantify the affinities of these compounds to various nucleic acids, including the RRE, tRNA, and duplex DNA. All the modified aminoglycosides exhibit a high affinity for the Rev binding site on the RRE (K(d) 相似文献   

Trigonostemons G and H,dinorditerpenoid dimers with axially chiral biaryl linkage from Trigonostemon chinensis

Trigonostemons G and H, two novel dimeric dinorditerpenoids, were isolated from the stem barks of Trigonostemon chinensis. Their planar structures and relative configurations were established by extensive analysis of spectroscopic data. Trigonostemons G and H possess a homodimeric biaryl skeleton obtained from two rearranged chiral nonracemic abietane-type dinorditerpenes through an axially chiral biaryl 11,11′-linkage. Torsional scan and computation of the transition states were carried out to estimate the rotational energy barrier, and the axial chirality (aS) was determined by time-dependent density functional theory (TDDFT) electronic circular dichroism (ECD) calculations. The positive n-π* ECD transitions of the isolated carbonyl chromophore above 300 nm could be used to determine the central chirality of trigonostemon G independently by ECD calculations of the diastereomers.     

Template specificity changes of DNA-dependent RNA polymerase in B. subtilis during sporulation

Previous work has indicated that loss of ability of DNA dependent RNA polymerase, from stationary phase cultures of $\text{B. subtilis}$, to transcribe phage øe DNA was a $\text{sine qua non}$ for sporulation. To ascertain if this change in template specificity was sporulation-specific, we repeated these experiments using a defined sporulation medium. The changes observed previously did not occur in the defined medium although sporulation was normal. The ability of the enzyme to transcribe other DNA templates was also examined. Similar studies were carried out using a polymerase from a rifamycin-resistant, sporulation conditional mutant. The significance of these findings with regard to the regulation of sporulation in $\text{B. subtilis}$ is discussed.     

A constitutively active GPCR retains its G protein specificity and the ability to form dimers

G protein-coupled receptors (GPCRs) are cell surface proteins which help to regulate the physiology of all the major organ systems within higher eukaryotes. They are stimulated by multiple ligands and activate a range of effector molecules to bring about changes in cell behaviour. The use of constitutively active mutants (CAMs) of GPCRs has enabled a better understanding of receptor activation as CAMs exhibit ligand-independent signalling negating the use of ligands. Here we introduce the fission yeast Schizosaccharomyces pombe as a host for producing CAMs, by describing the isolation and characterization of constitutive mutants of the P-factor receptor (Mam2). One mutant Mam2[P261L] contained a single-amino-acid substitution (Pro261 to Leu) within a region of high homology in GPCRs. Substitution of this proline leads to an 18-fold increase in ligand-independent signalling. We utilized Mam2[P261L] to investigate CAM activity by demonstrating that Mam2[P261L] is efficiently trafficked to the cell surface where it can form fully functional oligomeric complexes with the native receptor. Mam2[P261L] also retains the G protein specificity (RG-profile) of the native receptor and only induces constitutive signalling in the same G proteins. Finally, evidence is provided to indicate that CAM activity results from a reduction in the kinetics of G protein binding. This is the first time that S. pombe has been utilized for isolating and characterizing CAMs and the techniques employed will complement the current systems available for studying these important receptors.